Inhibition of the Expression of the Small Heat Shock Protein αB-Crystallin Inhibits Exosome Secretion in Human Retinal Pigment Epithelial Cells in Culture

Abstract

Exosomes carry cell type-specific molecular cargo to extracellular destinations and therefore act as lateral vectors of intercellular communication and transfer of genetic information from one cell to the other. We have shown previously that the small heat shock protein ␣B-crystallin (␣B) is exported out of the adult human retinal pigment epithelial cells (ARPE19) packaged in exosomes. Here, we demonstrate that inhibition of the expression of ␣B via shRNA inhibits exosome secretion from ARPE19 cells indicating that exosomal cargo may have a role in exosome biogenesis (synthesis and/or secretion). Sucrose density gradient fractionation of the culture medium and cellular extracts suggests continued synthesis of exosomes but an inhibition of exosome secretion. In cells where ␣B expression was inhibited, the distribution of CD63 (LAMP3), an exosome marker, is markedly altered from the normal dispersed pattern to a stacked perinuclear presence. Interestingly, the total anti-CD63(LAMP3) immunofluorescence in the native and ␣B-inhibited cells remains unchanged suggesting continued exosome synthesis under conditions of impaired exosome secretion. Importantly, inhibition of the expression of ␣B results in a phenotype of the RPE cell that contains an increased number of vacuoles and enlarged (fused) vesicles that show increased presence of CD63(LAMP3) and LAMP1 indicating enhancement of the endolysosomal compartment. This is further corroborated by increased Rab7 labeling of this compartment (RabGTPase 7 is known to be associated with late endosome maturation). These data collectively point to a regulatory role for ␣B in exosome biogenesis possibly via its involvement at a branch point in the endocytic pathway that facilitates secretion of exosomes.Exosomes are 50 -200-nm nanovesicles produced via the endosomal pathway of protein homeostasis (1-3). The process of endocytosis and the trafficking of endocytosed vesicles to lysosomes are major pathways of protein metabolism that regulate transmembrane protein (receptor) turnover at the plasma membrane. The exosome biogenesis starts with the early endosome that matures into a late endosome concomitant with its intraluminal vesicles, making it a multivesicular body (MVB). 2The MVB is at a branch point of this pathway from where it may progress toward two different cellular compartments with two entirely different physiological consequences. The first one is that the MVB may fuse with the lysosomes (generating the endolysosomal compartment) leading to the degradation of its vesicular contents; the second is that the MVB traffics to and fuses with the plasma membrane, leading to the release of its intraluminal vesicles as exosomes carrying the active macromolecular cargo. What dictates either of the two fates is not known (4).RPE is an important single layer of cells at the interface of the blood-retina barrier. It provides critical physiological support for the maintenance of the photoreceptor neurons (5, 6). It is a polarized epithelium, which on its apical side nourishes the ...

Preclinical stress originates in the rat optic nerve head during development of autoimmune optic neuritis

“…In order to further our understanding regarding the function of αB-crystallin upregulation, we next used shRNA to knock-down its expression. Treatment of oligodendrocyte cultures with lentiviral particles carrying shRNA constructs previously demonstrated to reduce αB-crystallin expression (Gangalum, Bhat, Kohan, & Bhat, 2016) similarly reduced expression of αB-crystallin in cultures which were treated with EAE sera (Figure 6k). Cell survival of lentiviral-transfected oligodendrocyte cultures was then assayed following exposure to sera.…”

Extracellular vesicle-mediated long-range communication in stressed retinal pigment epithelial cell monolayers

“…In early AMD, the RPE is thought to contribute to the formation of drusen (extracellular material that builds up between the RPE and Bruch’s membrane). Wang et al, had proposed that exosomes released from aged RPE might contribute to the formation of drusen [50], and polarized release of exosomes from RPE cells has been described in cultured RPE cells [39, 82, 83]. In addition to damaging effects, exosomes released from the RPE are also thought to contribute to neuroptrotection in the retina [84].…”

Roles of exosomes in the normal and diseased eye

“…In addition, both heparanase and syndecan-1 are involved in exosome biogenesis and regulation of exosome release (Thompson et al, 2013). Furthermore, a recent study by Gangalum and colleagues (Gangalum et al, 2016) reported that shRNA knockdown of αB-Crystallin in ARPE-19 cells severely inhibits both apical and basolateral exosome and/or small EV release. Thus, approaches attempting to regulate exosome release in the affected cell types, as well as targeting proteoglycans found on exosomes and in the ECM of the retinal vasculature and BrM at the choriocapillarismay be able to reduce neovascularization in neovascular AMD and diabetic retinopathy.…”

“…An exhaustive characterization of EVs released both apically and basolaterally from RPE cells under normal and pathophysiological conditions is critical to elucidating the potential role of exosomes in the AMD disease process. Interestingly, a recent study showed that under serum-free conditions of exosome collection from polarized ARPE-19 cultures, the total exosome release was about twofold higher on the apical vs basolateral side (Gangalum et al, 2016). Our own studies using polarized primary RPE cultures show similar results under FBS-supplemented culture conditions.…”